This application is a national stage filing under 35 U.S.C. 371 of PCT application PCT/GB99/02801, filed Aug. 25, 1999, which claims priority from European Application Nos. 98402144.4, filed Aug. 31, 1998, and 99401351.4, filed Jun. 4, 1999, the specifications of all of which are incorporated by reference herein. PCT Application PCT/GB99/02801 was published under PCT Article 21(2) in English.
The present invention relates to compounds useful in the inhibition of metalloproteinases and in particular to pharmaceutical compositions comprising these, as well as their use.
The compounds of this invention are inhibitors of one or more metalloproteinase enzymes. Metalloproteinases are a superfamily of proteinases (enzymes) whose numbers in recent years have increased dramatically. Based on structural and functional considerations these enzymes have been classified into families and subfamilies as described in N. M. Hooper (1994) FEBS Letters 354:1-6. Examples of metalloproteinases include the matrix metalloproteinases (MMP) such as the collagenases (MMP1, MMP8, MMP13), the gelatinases (MMP2, MMP9), the stromelysins (MMP3, MMP10, MMP11), matrilysin (MMP7), metalloelastase (MMP12), enamelysin (MMP19), the MT-MMPs (MMP14, MMP15, MMP16, MMP17); the reprolysin or adamalysin or MDC family which includes the secretases and sheddases such as TNF converting enzymes (ADAM10 and TACE); the astacin family which include enzymes such as procollagen processing proteinase (PCP), and other metalloprotenases such as aggrecanase, the endothelin converting enzyme family and the angiotensin converting enzyme family.
Metalloproteinases are believed to be important in a plethora of physiological disease processes that involve tissue remodelling such as embryonic development, bone formation and uterine remodelling during menstruation. This is based on the ability of the metalloproteinases to cleave a broad range of matrix substrates such as collagen, proteoglycan and fibronectin. Metalloproteinases are also believed to be important in the processing, or secretion, of biological important cell mediators, such as tumour necrosis factor (TNF); and the post translational proteolysis processing, or shedding, of biologically important membrane proteins, such as the low affinity IgE receptor CD23 (for a more complete list see N. M. BHooper et al., (1997) Biochem J. 321 265-279).
Metalloproteinases have been associated with many disease conditions. Inhibition of the activity of one or more metalloproteinases may well be of benefit in these disease conditions, for example: various inflammatory and allergic diseases such as, inflammation of the joint (especially rheumatoid arthritis, osteoarthritis and gout), inflammation of the gastro-intestinal tract (especially inflammatory bowel disease, ulcerative colitis and gastritis), inflammation of the skin (especially psoriasis, eczema, dermatitis); in tumour metastasis or invasion; in disease associated with uncontrolled degradation of the extracellular matrix such as osteoarthritis; in bone resorptive disease (such as osteoporosis and Paget""s disease)); in diseases associated with aberrant angiogenesis; the enhanced collagen remodelling associated with diabetes, periodontal disease (such as gingivitis), corneal ulceration, ulceration of the skin, post-operative conditions (such as colonic anastomosis) and dermal wound healing; demyelinating diseases of the central and peripheral nervous systems (such as multiple sclerosis); Alzheimer""s disease; and extracellular matrix remodelling observed in cardiovascular diseases such as restenosis and atheroscelerosis.
A number of metalloproteinase inhibitors are known; different classes of compounds may have different degrees of potency and selectivity for inhibiting various metalloproteinases. We have discovered a new class of compounds that are inhibitors of metalloproteinases and are of particular interest in inhibiting MMP-13, as well as MMP-9. The compounds of this invention have beneficial potency and/or pharmacokinetic properties.
MMP13, or collagenase 3, was initially cloned from a cDNA library derived from a breast tumour [J. M. P. Freije et al. (1994) Journal of Biological Chemistry 269(24):16766-16773]. PCR-RNA analysis of RNAs from a wide range of tissues indicated that MMP13 expression was limited to breast carcinomas as it was not found in breast fibroadenomas, normal or resting mammary gland, placenta, liver, ovary, uterus, prostate or parotid gland or in breast cancer cell lines (T47-D, MCF-7 and ZR75-1). Subsequent to this observation MMP13 has been detected in transformed epidermal keratinocytes [N. Johansson et al., (1997) Cell Growth Differ. 8(2):243-250], squamous cell carcinomas [N. Johansson et al., (1997) Am. J. Pathol. 151(2):499-508] and epidermal tumours [K. Airola et al., (1997) J. Invest. Dermatol. 109(2):225-231]. These results are suggestive that MMP13 is secreted by transformed epithelial cells and may be involved in the extracellular matrix degradation and cell-matrix interaction associated with metastasis especially as observed in invasive breast cancer lesions and in malignant epithelia growth in skin carcinogenesis.
Recent published data implies that MMP13 plays a role in the turnover of other connective tissues. For instance, consistent with MMP13""s substrate specificity and preference for degrading type II collagen [P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761-768; V. Knauper el al., (1996) The Biochemical Journal 271: 1544-1550], MMP13 has been hypothesised to serve a role during primary ossification and skeletal remodelling [M. Stahle-Backdahl et al., (1997) Lab. Invest. 76(5):717-728; N. Johansson et al., (1997) Dev. Dyn. 208(3):387-397], in destructive joint diseases such as rheumatoid and osteo-arthritis [D. Wernicke et al., (1996) J. Rheumatol. 23:590-595; P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3:761-768; O. Lindy et al., (1997) Arthritis Rheum 40(8):1391-1399]; and during the aseptic loosening of hip replacements [S. Imai et al, (1998) 3. Bone Joint Surg. Br. 80(4):701-710]. MMP13 has also been implicated in chronic adult periodontitis as it has been localised to the epithelium of chronically inflamed mucosa human gingival tissue [V. J. Uitto et al., (1998) Am. J. Pathol 152(6): 1489-1499] and in remodelling of the collagenous matrix in chronic wounds [M. Vaalamo et al., (1997) J. Invest. Dermatol. 109(l):96-101].
MMP9 (Gelatinase B; 92 kDa TypeIV Collagenase; 92 kDa Gelatinase) is a secreted protein which was first purified, then cloned and sequenced, in 1989 (S. M. Wilhelm et al (1989) J. Biol Chem. 264 (29): 17213-17221. Pubished erratum in J. Biol Chem. (1990) 265 (36): 22570.). A recent review of MMP9 provides an excellent source for detailed information and references on this protease: T. H. Vu and Z. Werb (1998) (In: Matrix Metalloproteinases. 1998. Edited by W. C. Parks and R. P. Mecham. pp 115-148. Academic Press. ISBN 0-12-545090-7). The following points are drawn from that review by T. H. Vu and Z. Werb (1998).
The expression of MMP9 is restricted normally to a few cell types, including trophoblasts, osteoclasts, neutrophils and macrophages. However, it""s expression can be induced in these same cells and in other cell types by several mediators, including exposure of the cells to growth factors or cytokines. These are the same mediators often implicated in initiating an inflammatory response. As with other secreted MMPs, MMP9 is released as an inactive Pro-enzyme which is subsequently cleaved to form the enzymatically active enzyme. The proteases required for this activation in vivo are not known. The balance of active MMP9 versus inactive enzyme is further regulated in vivo by interaction with TIMP-1 (Tissue Inhibitor of Metalloproteinases-1), a naturally-occurring protein. TIMP-1 binds to the C-terminal region of M9, leading to inhibition of the catalytic domain of MP9. The balance of induced expression of ProMMP9, cleavage of Pro- to active MMP9 and the presence of TIMP-1 combine to determine the amount of catalytically active MMP9 which is present at a local site. Proteolytically active MMP9 attacks substrates which include gelatin, elastin, and native Type IV and Type V collagens; it has no activity against native Type I collagen, proteoglycans or laminins. There has been a growing body of data implicating roles for MMP9 in various physiological and pathological processes. Physiological roles include the invasion of embryonic trophoblasts through the uterine epithelium in the early stages of embryonic implantation; some role in the growth and development of bones; and migration of inflammatory cells from the vasculature into tissues. Increased MMP9 expression has observed in certain pathological conditions, therebye implicating MMP9 in disease processed such as arthritis, tumour metastasis, Alzheimer""s, Multiple Sclerosis, and plaque rupture in atherosclerosis leading to acute coronary conditions such as Myocardial Infarction.
In a first aspect of the invention we provide compounds of the formula I 
wherein ring B is a monocyclic or bicyclic alkyl, aryl, aralkyl, heteroaryl or heteroaralkyl ring comprising up to 12 ring atoms and containing one or more heteroatoms independently chosen from N, O, and S; alternatively ring B may be biphenyl; ring B may optionally be linked to ring A by a C1-4 alkyl or a C1-4 alkoxy chain linking the 2-position of ring B with a carbon atom alpha to X2;
each R3 is independently selected from hydrogen, halogen, NO2, COOR wherein R is hydrogen or C1-6 alkyl, CN, CF3, C1-6 alkyl, xe2x80x94Sxe2x80x94C1-6 alkyl, xe2x80x94SOxe2x80x94C1-6 alkyl, xe2x80x94SO2xe2x80x94C1-6 alkyl , C1-6 alkoxy and up to C10 aryloxy, n is 1,2 or 3;
P is xe2x80x94(CH2)n- wherein n=0, 1, 2, or P is an alkene or alkyne chain of up to six carbon atoms; where X2 is C, P may be -Het-, xe2x80x94(CH[R6])n-Het-, -Het-(CH[R6]n- or -Het-(CH[R6])n-Het-, wherein Het is selected from xe2x80x94COxe2x80x94, xe2x80x94Sxe2x80x94, SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR6-, or xe2x80x94Oxe2x80x94 wherein n is 1 or 2, or P may be selected from xe2x80x94COxe2x80x94N(R6)xe2x80x94, xe2x80x94N(R6)xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94N(R6)xe2x80x94 and xe2x80x94N(R6)xe2x80x94SO2xe2x80x94, and R6 is hydrogen, C1-6 alkyl up to C10 aralkyl or up to C9 heteroalkyl;
Ring A is a 5-7 membered aliphatic ring and may optionally be mono- or di-substituted by optionally substituted C1-6 alkyl or C1-6 alkoxy, each substituent being independently selected from halogen, C1-6 alkyl or an oxo group,
X1 and X2 are independently selected from N and C, where a ring substituent on ring A is an oxo group this is preferably adjacent a ring nitrogen atom;
Y is selected from xe2x80x94SO2xe2x80x94and xe2x80x94COxe2x80x94;
Z is xe2x80x94CONHOH, Y is xe2x80x94COxe2x80x94 and Q is selected from xe2x80x94C(R6)(R7)xe2x80x94, CR6)(R7)xe2x80x94CH2xe2x80x94, xe2x80x94N(R6)xe2x80x94, and xe2x80x94N(R6)xe2x80x94CH2xe2x80x94 wherein R6 is as defined above, and solely in relation to Q as here defined, R6 may also represent up to C10 aryl and up to C9 heteroaryl, and R7 is H, C1-6 alkyl, or together with R6 forms a carbocyclic or heterocyclic spiro 5, 6 or 7 membered ring, the latter containing at least one heteroatom selected from N, O, and S;
Z is xe2x80x94CONHOH, Y is xe2x80x94SO2xe2x80x94 and Q is selected from xe2x80x94C(R6)(R7)xe2x80x94, and xe2x80x94C(R6)(R7)xe2x80x94CH2xe2x80x94;
or Z is xe2x80x94N(OH)CHO and Q is selected from xe2x80x94CH(R6)xe2x80x94, xe2x80x94CH(R6)xe2x80x94CH2xe2x80x94, and xe2x80x94N(R6)xe2x80x94CH2xe2x80x94;
R1 is H, C1-6 alkyl, C5-7 cycloalkyl, up to C10 aryl, up to C10 heteroaryl, up to C12 aralkyl, or up to C12 heteroarylalkyl, all optionally substituted by up to three groups independently selected from NO2, CF3, halogen, C1-4 alkyl, carboxy(C1-4)alkyl, up to C6 cycloalkyl,xe2x80x940R4, xe2x80x94SR4, C1-4 alkyl substituted with xe2x80x94OR4, SR4 (and its oxidised analogues), NR4, Nxe2x80x94Yxe2x80x94R4, or C1-4 alkyl-Yxe2x80x94NR4, with the proviso that where R1 is xe2x80x94OH, xe2x80x94OR4, xe2x80x94SR4, or NR4, or Nxe2x80x94Yxe2x80x94R4 then Z is not xe2x80x94N(OH)CHO, or R1 is 2,3,4,5,6-pentafluorophenyl;
R4 is hydrogen, C1-6 alkyl, up to C10 aryl or up to C10 heteroaryl or up to C9 aralkyl, each independently optionally substituted by halogen, NO2, CN, CF3, C1-6 alkyl, xe2x80x94Sxe2x80x94C1-6 alkyl, xe2x80x94SOxe2x80x94C1-6 alkyl, xe2x80x94SO2xe2x80x94C1-6 alkyl or C1-6 alkoxy;
R2 is H, C1-6 alkyl, or together with R1 forms a carbocyclic or heterocyclic spiro 5, 6 or 7 membered ring, the latter containing at least one heteroatom selected from N, O, and S;
also the group Q can be linked to either R1 or R2 to form a 5, 6 or 7 membered alkyl or heteroalkyl ring comprising one or more of O, S and N.
Any alkyl groups outlined above may be straight chain or branched.
Convenient values for the above groups include the following:
ring A=a 5-6 membered aliphatic ring, such as a piperazine ring, and may optionally be mono- or di-substituted by optionally substituted C1-6 alkyl or C1-6-alkoxy, each substituent being independently selected from halogen, C1-6 alkyl or an oxo group;
R3=hydrogen, halogen, NO2, CF3, C1-4 alkyl, and C1-4 alkoxy, n is 1 or 2, such as individually 4-fluoro, CF3, 4-chloro and 3,4-dichloro;
ring B=monocyclic or bicyclic aryl, aralkyl or heteroaryl having up to 10 ring atoms, especially monocyclic aryl, aralkyl or heteroaryl having up to 7 ring atoms, more especially monocyclic aryl or heteroaryl having up to 6 ring atoms, such as a phenyl or pyridyl ring;
P=xe2x80x94(CH2)n- wherein n is 0 or 1, or xe2x80x94Oxe2x80x94, or xe2x80x94COxe2x80x94N(R6)xe2x80x94;
one or both of X2 and X1=N, or X1 is N, or X2 is C;
Y=xe2x80x94SO2xe2x80x94, Y=xe2x80x94COxe2x80x94;
Q=xe2x80x94CH(R6)xe2x80x94, xe2x80x94CH(R6)xe2x80x94CH2xe2x80x94, and xe2x80x94N(R6)xe2x80x94CH2xe2x80x94 wherein R6 is hydrogen or C1-6 alkyl; also where Q is linked to R1 or R2 to form a C5-7 alkyl or heteroalkyl ring such as a cyclohexyl ring;
R1 hydrogen, C1-6 alkyl, C5-7 cycloalkyl, up to C12 aralkyl, up to C11 heteroarylalkyl, up to C10 aryl or heteroaryl such as up to C6 aryl; all optionally substituted by up to three halogen atoms, or by CF3;
R2=hydrogen, or together with R1 represent a carbocyclic or heterocyclic spiro 5- or 6 membered ring, such as a tetrahydropyran ring;
R4 up to C10 aryl optionally substituted by halogen, NO2, CN, CF3, C1-6 alkyl, xe2x80x94Sxe2x80x94C1-6 alkyl, xe2x80x94SOxe2x80x94C1-6 alkyl, xe2x80x94SO2xe2x80x94C1-6 alkyl or C1-6 alkoxy;
Z=xe2x80x94CONHOHxe2x80x94, Z=xe2x80x94N(OH)CHO.
Preferred values for the above groups include the following:
R3=hydrogen, halogen such as chlorine, bromine or fluorine, NO2, CF3, methyl, ethyl, methoxy, ethoxy, particularly methoxy or fluorine;
ring B=a monocyclic aryl, aralkyl or heteroaryl ring having up to 7 ring atoms such as phenyl, biphenyl, napthyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, especially phenyl, pyridyl and pyrimidyl, more especially phenyl, 2-pyridyl and 2,4-pyrimidyl;
P=a direct bond;
both X2 and X1 are N;
Y=xe2x80x94SO2xe2x80x94;
Q=xe2x80x94CH2xe2x80x94;
R1 is phenyl, 4-trifluoromethylphenyl, phenethyl, phenpropyl, isobutyl, cyclopentyl, benzyloxymethyl, 3,4-dichlorophenyl, pyridyl, pyridylethyl, thiophenylpropyl, bromothiophenyl, pyrimidinylethyl, pyrimidinylpropyl, pyridylethyl, pyridylpropyl or together with R2 is spirocyclohexane or spiro-4-pyran; R2 is hydrogen
Z=xe2x80x94N(OH)CHO.
More preferred values include R3 being halogen, the substituent is preferably meta or para to the ring junction where ring B is an aryl or heteroaryl ring, where ring B is phenyl then especially 4-fluoro and where ring B is pyridyl then 3-, or 4-chloro (as appropriate);
Q=xe2x80x94CH2xe2x80x94.
Preferred combinations of Rings B and A include phenyl and piperazinyl; pyridyl and piperazinyl, and pyrimidine and piperazinyl respectively.
Particular alicyclic, fused and heterocyclic rings for ring B include any one of 
Particular rings for ring A include any one of 
and its corresponding seven membered analogue(s).
It will be appreciated that the particular substitituents and number of substituents on rings A and B are selected so as to avoid sterically undesirable combinations. This also applies to rings as may be formed by R1 and Q, R2 and Q as well as R6 and R7.
Where optically active centres exist in the compounds of formula I, we disclose all individual optically active forms and combinations of these as individual specific embodiments of the invention, as well as their corresponding racemates.
Specific compounds include 
wherein R=phenyl or phenethyl and 
wherein R=isobutyl or a spiro4-pyran ring
As previously outlined the compounds of the invention are metalloproteinase inhibitors, in particular they are inhibitors of MMP13. Each of the above indications for the compounds of the formula I represents an independent and particular embodiment of the invention. Whilst we do not wish to be bound by theoretical considerations, the compounds of the invention are believed to show selective inhibition for any one of the above indications relative to any MMP1 inhibitory activity, by way of non-limiting example they may show 100-1000 fold selectivity over any MMP1 inhibitory activity.
In addition we have found that compounds of the formula 1 wherein ring B is phenyl, pyridyl (such as 2-pyridyl or 3-pyridyl, especially 2-pyridyl) ring optionally mono- or di-substituted, preferably mono-substituted, by halogen (for example chlorine ), P is a direct bond; ring A is a piperazinyl or piperidinyl ring, Y is xe2x80x94SO2xe2x80x94 and Q is C1-4 alkylene (for example xe2x80x94CH2xe2x80x94), especially xe2x80x94CH2xe2x80x94; R1 is as defined for Formula 1 and is especially 2-phenylpropyl, 2-(2pyridyl)propyl, 2-(3-pyridyl)propyl, 2-(4-pyridyl)propyl, phenyl, benzyloxymethyl, 4-phenylbutyl, 2-phenylbutyl, or 2-(2thienyl)propyl; and Z is xe2x80x94N(OH)CHO; are of particular use as aggrecanase inhibitors ie. inhibitors of aggrecan degradation. Of particular note are compounds of the formula I wherein ring B is a phenyl, 3-methylphenyl, 4-fluorophenyl, 3-chrorophenyl, 4-chlorophenyl, or 3,4-dichyorophenyl ring or 5-chloro-2-pyridyl; P is a direct bond, ring A is piperidinyl or piperazinyl especially piperazinyl, Y is SO2, Q is xe2x80x94CH2xe2x80x94, Z is xe2x80x94N(OH)CHO and R1 is phenyl, phenbutylene, phenisopropylene, 2-pyridylethylene, 2-pyridylisopropylene, 3-pyridylisopropylene, 4-pyridylisopropylene, or 4chlorophenyloxydimethylmethylene. Also of mention are compounds of the formula I wherein ring B is phenyl monosubstituted by chlorine or fluorine, especially 4-chlorophenyl and 4-fluorophenyl; P is a direct bond; ring A is piperidinyl, Y is SO2, Q is xe2x80x94CH2xe2x80x94, Z is xe2x80x94CONHOH and R1 is hydrogen, i-butyl, or spiro-tetrahydropyranyl.
Particular compounds include
wherein PIP=piperazinyl
RH=reverse hydroxamate group
and R2=hydrogen
The compounds of the invention may be provided as pharmaceutically acceptable salts. These include acid addition salts such as-hydrochloride, hydrobromide, citrate and maleate salts and salts formed with phosphoric and sulphuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium or potassium, an alkaline earth metal salt for example calcium or magnesium, or organic amine salt for example triethylamine.
They may also be provided as in vivo hydrolysable esters. These are pharmaceutically acceptable esters that hydrolyse in the human body to produce the parent compound. Such esters can be identified by administering, for example intravenously to a test animal, the compound under test and subsequently examining the test animal""s body fluids. Suitable in vivo hydrolysable esters for carboxy include methoxymethyl and for hydroxy include formyl and acetyl, especially acetyl.
In order to use a compound of the formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Therefore in another aspect the present invention provides a pharmaceutical composition which comprises a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester and pharmaceutically acceptable carrier.
The pharmaceutical compositions of this invention may be administered in standard manner for the disease condition that it is desired to treat, for example by oral, topical, parenteral, buccal, nasal, vaginal or rectal adminstration or by inhalation. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.
In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain, or be co-administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more disease conditions referred to hereinabove.
The pharmaceutical compositions of this invention will normally be administered to humans so that, for example, a daily dose of 0.5 to 75 mg/kg body weight (and preferably of 0.5 to 30 mg/kg body weight) is received. This daily dose may be given in divided doses as necessary, the precise amount of the compound received and the route of administration depending on the weight, age and sex of the patient being treated and on the particular disease condition being treated according to principles known in the art.
Typically unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.
Therefore in a further aspect, the present invention provides a compound of the formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof for use in a method of therapeutic treatment of the human or animal body.
In yet a further aspect the present invention provides a method of treating a metalloproteinase mediated disease condition which comprises administering to a warm-blooded animal a therapeutically effective amount of a compound of the formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof
In another aspect the present invention provides a process for preparing a compound of the formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof which process comprises
a) reacting a compound of the formula (II) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof with a compound of the formula (III) 
wherein X1I represents X or a precursor of X (whether by modification or displacement) or an activated form of X suitable for reaction with Y1;
Y1 represents Y, a precursor of Y, or an activated form of Y suitable for reaction with X1I; by way of non-limiting example, when X is C then X1 may be derivatised to include a precursor of Y for reaction with a compound of formula III wherein YI is a precursor of Y;
ZI represents a protected form of Z, a precursor of Z (whether by modification or displacement of ZI) or an activated form of Z;
and where Q=xe2x80x94CH2)(R6)xe2x80x94 then by reacting a compound of the formula IX with an appropriate compound of the formula R1-COxe2x80x94R2 to yield an alkene of the formula X, which is then converted to a compound of the formula XI wherein Z* is a hydroxylamine precursor of the group Z, and then converting Z* to the group Z, all as set out below: 
or
b) reacting a compound of the formula (IV) ) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof with a compound of the formula (V). 
wherein BI represents a suitable ring function or substituent group for reaction with PI;
ZI is as hereinbefore defined; and
Pi represents a suitably activated form of the linker P for reaction with BI 
or where X2=N then P1 may be present on ring A rather than ring B
or, as required, the linker P may be formed by appropriate reaction of precursor groups Pxe2x80x3 and Pxe2x80x2xe2x80x3 provided on rings BI and A respectively, or vice versa.
A compound of the formula (II) is conveniently prepared by reacting a compound of the formula (VI) with a compound of the formula (VII) 
wherein BI represents a suitable ring function or substituent group, X2I represents X or a precursor of X (whether by modification or displacement) or an activated form of X suitable for reaction with BI and wherein BI and X2I when reacted together provide the linker P between ring A and ring B in the compound of formula (II). By way of non-limiting example, when X2 is N then ring B is suitably derivatised to introduce the linker P via BI, and when X2 is C then both ring B and ring A are suitably derivatised to provide the linker P by the reaction of BI and X2I.
It will be appreciated that many of the relevant starting materials are commercially available. In addition the following table shows details of aldehyde intermediates and their corresponding registry numbers in Chemical Abstracts.
3-(2-pyrimidyl) propionaldehyde. To a solution of 2-Bromopyrimidine (7.95 g, 0.05 M) in acetonitrile (150 mL) was added propargylalcohol (4.2 g, 0.075 M ), bis-(triphenylphosphine)-palladium(11)chloride (750 mg, 1 mM), copper iodide (100 mg, 0.5 mM) and triethylamine (25 mL, 0.25 M) and the mixture was stirred and heated at 70xc2x0 C. for 2 hours. An additional amount of propargyl alcohol (2.1 g, 0.038 M), bis-(triphenylphosphine)-palladium(11)chloride (375 mg, 0.5 mil), and copper iodide (50 mg, 0.25 mil) was then added to the reaction mixture which was stirred and heated at 70xc2x0 C. for an additional 1 hour.
The reaction mixture was evaporated to dryness and the residue which was pre-adsorbed on to silica, chromatographed. Elution with ethyl acetate gave 3-(2-pyrimidyl) prop-2-yn-3-ol as a yellow solid 4.45 g (66%). NMR (CDCl3) 2.9 (1H, t), 4.5 (2H, d), 7.3 (1H, d), 8.8 (2H, t), MS found MH+ 135.
3-(2-pyrimidyl)propan-1-ol (4.45 g; 0.033 M) was dissolved in ethyl acetate (140 mL), 10% Pd/C (890 mg) was added and the mixture stirred under an atmosphere of hydrogen for 6 hours. The reaction mixture was filtered through Celite and the filtrate evaporated to give 3-(2-pyrimidyl)propan-1-ol as a yellow oil, 4.15 g (91%). NMR (CDCl3) 2.1 (2H, m), 3.2 (2H, t), 3.8 (2H, t), 7.2 (1H, t), 8.7 (2H, d) MS found MH+ 139.
3-(2-pyrimidyl)propan-1-ol was oxidized to give 3-(2-pyrimidyl) propionaldehyde as a yellow oil NMR (CDCl3) 3.0 (2H, t), 3.4 (2H, t), 7.1 (1H, t), 8.7 (2H, d), 9.9 (1H, s) using the Swern oxidation described in this patent.
Using the procedure described above the following aldehydes were prepared 4-(2-pyrimidyl) butyraldehyde by using 3-butyn-1-ol in place of propargylalcohol.
NMR CDCl3 9.8 (1H, s), 8.6 (2H, m), 7.15 (1H, m), 3.0 (2H, m), 2.5 (2H, m), 2.2 (2H, m).
4-(5-pyrimidyl)butyraldehyde by using 3-butyn-1-ol in place of propargylalcohol and 5-bromopyrirnidine in place of 2-bromopyrimidine NMR CDCl3 9.8 (1H, s), 9.1 (1H, s), 8.6 (2h, s), 2.7 (2H, t), 2.55 (2H, t), 2.0 (2H, m).
4-(2-pyridyl)butyraldehyde by using 3-butyn-1-ol in place of propargylalcohol and 2-bromopyridine in place of 2-bromopyrimidine.
NMR CDCl3 9.8 (1H, s); 8.6 (1H, d), 7.6 (1H, m); 7.1 (2H, m) 2.8 (2H, t), 2.55 (2H, t), 2.0 (2H, m).
The compounds of the invention may be evaluated for example in the following assays:
Matrix Metalloproteinase family including for example MMP13.
Recombinant human proMMP13 may be expressed and purified as described by Knauper et al. [V. Knauper et al., (1996) The Biochemical Journal 271:1544-1550 (1996)]. The purified enzyme can be used to monitor inhibitors of activity as follows: purified proMMP13 is activated using 1 mM amino phenyl mercuric acid (APMA), 20 hours at 21xc2x0 C.; the activated MMP13 (11.25 ng per assay) is incubated for 4-5 hours at 35xc2x0 C. in assay buffer (0.1M Tris-HCl, pH 7.5 containing 0.1M NaCl, 20 mM CaCl2, 0.02 mM ZnCl and 0.05% (w/v) Brij 35 using the synthetic substrate 7-methoxycoumarin-4-yl)acetyl.Pro.Leu.Gly.Leu.N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl.Ala.Arg.NH2 in the presence or absence of inhibitors. Activity is determined by measuring the fluorescence at xcexex 328 nm and xcexem393 nm. Percent inhibition is calculated as follows: % Inhibition is equal to the [Fluorescenceplus inhibitorxe2x88x92Fluorescencebackground] divided by the [Fluorescenceminus inhibitorxe2x88x92Fluorescencebackground].
A similar protocol can be used for other expressed and purified pro MMPs using substrates and buffers conditions optimal for the particular MMP, for instance as described in C. Graham Knight el al., (1992) FEBS Lett. 296(3):263-266
The ability of the compounds to inhibit proTNFxcex1 convertase enzyme may be assessed using a partially purified, isolated enzyme assay, the enzyme being obtained from the membranes of THP-1 as described by K. M. Mohler et al., (1994) Nature 370:218-220. The purified enzyme activity and inhibition thereof is determined by incubating the partially purified enzyme in the presence or absence of test compounds using the substrate 4xe2x80x2,5xe2x80x2-Dimethoxy-fluoresceinyl Ser.Pro.Leu.Ala.Gln.Ala.Val.Arg.Ser.Ser.Ser.Arg.Cys(4-(3-succinimid-1-yl)-fluorescein)-NH2 in assay buffer (50 mM Tris HCl, pH 7.4 containing 0.1% (w/v) Triton X-100 and 2 mM CaCl2), at 26xc2x0 C. for 18 hours. The amount of inhibition is determined as for MMP13 except xcexex 490 nm and xcexem 530 nm were used. The substrate was synthesised as follows. The peptidic part of the substrate was assembled on Fmoc-NH-Rink-MBHA-polystyrene resin either manually or on an automated peptide synthesiser by standard methods involving the use of Fmoc-amino acids and O-benzotriazol-1-yl-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (HBTU) as coupling agent with at least a 4- or 5-fold excess of Fmoc-amino acid and HBTU. Ser1 and Pro2 were double-coupled. The following side chain protection strategy was employed; Ser1(But), Gln5(Trityl), Arg8,12(Pmc or Pbf), Ser9,10,11(Trityl), Cys13(Trityl). Following assembly, the N-terminal Fmoc-protecting group was removed by treating the Fmoc-peptidyl-resin with in DMF. The amino-peptidyl-resin so obtained was acylated by treatment for 1.5-2 hr at 70xc2x0 C. with 1.5-2 equivalents of 4xe2x80x2,5xe2x80x2-dimethoxy-fluorescein-4(5)-carboxylic acid [Khanna and Ullman, (1980) Anal Biochem. 108:156-161) which had been preactivated with diisopropylcarbodiimide and 1-hydroxybenzotriazole in DMF]. The dimethoxyfluoresceinyl-peptide was then simultaneously deprotected and cleaved from the resin by treatment with trifluoroacetic acid containing 5% each of water and triethylsilane. The dimethoxyfluoresceinyl-peptide was isolated by evaporation, trituration with diethyl ether and filtration. The isolated peptide was reacted with 4-(N-maleimido)-fluorescein in DMF containing diisopropylethylamine, the product purified by RP-HPLC and finally isolated by freeze-drying from aqueous acetic acid. The product was characterised by MALDI-TOF MS and amino acid analysis.
The activity of the compounds of the invention as inhibitors of aggrecan degradation may be assayed using methods for example based on the disclosures of E. C. Arner et al., (1998) Osteoarthritis and Cartilage 6:214-228; (1999) Journal of Biological Chemistry, 274 (10), 6594-6601 and the antibodies described therein. The potency of compounds to act as inhibitors against collagenases can be determined as described by T. Cawston and A. Barrett (1979) Anal. Biochem. 99:340-345.
The ability of the compounds of this invention to inhibit the cellular processing of TNFxcex1 production may be assessed in THP-1 cells using an ELISA to detect released TNF essentially as described K. M. Mohler et al., (1994) Nature 370:218-220. In a similar fashion the processing or shedding of other membrane molecules such as those described in N. M. Hooper et al., (1997) Biochem. J. 321:265-279 may be tested using appropriate cell lines and with suitable antibodies to detect the shed protein.
The ability of the compound of this invention to inhibit the migration of cells in an invasion assay may be determined as described in A. Albini et al., (1987) Cancer Research 47:3239-3245.
The ability of the compounds of this invention to inhibit TNFxcex1 production is assessed in a human whole blood assay where LPS is used to stimulate the release of TNFxcex1. Heparinized (10 Units/ml) human blood obtained from volunteers is diluted 1:5 with medium (RPMI1640 +bicarbonate, penicillin, streptomycin and glutamine) and incubated (160 xcexcl) with 20 xcexcl of test compound (triplicates), in DMSO or appropriate vehicle, for 30 min at 37xc2x0 C. in a humidified (5% CO2/95% air) incubator, prior to addition of 20 xcexcl LPS (E. coli. 0111:B4; final concentration 10 xcexcg/ml). Each assay includes controls of diluted blood incubated with medium alone (96 wells/plate) or a known TNFxcex1 inhibitor as standard. The plates are then incubated for 6 hours at 37xc2x0 C. (humidified incubator), centrifuiged (2000 rpm for 10 min; 4xc2x0 C.), plasma harvested (50-100 xcexcl) and stored in 96 well plates at xe2x88x9270xc2x0 C. before subsequent analysis for TNFxcex1 concentration by ELISA.
The ability of the compounds of this invention to inhibit the degradation of the aggrecan or collagen components of cartilage can be assessed essentially as described by K. M. Bottomley e al., (1997) Biochem J. 323:483-488.
To evaluate the clearance properties and bioavailability of the compounds of this invention an ex vivo pharmacodynamic test is employed which utilises the synthetic substrate assays above or alternatively HPLC or Mass spectrometric analysis. This is a generic test which can be used to estimate the clearance rate of compounds across a range of species. Animals (e.g. rats, marmosets) are dosed iv or po with a soluble formulation of compound (such as 20% w/v DMSO, 60% w/v PEG400) and at subsequent time points (e.g. 5, 15, 30, 60, 120, 240, 480, 720, 1220 mins) the blood samples are taken from an appropriate vessel into IOU heparin. Plasma fractions are obtained following centrifugation and the plasma proteins precipitated with acetonitrile (80%w/v final concentration). After 30 mins at xe2x88x9220xc2x0 C. the plasma proteins are sedimented by centrifugation and the supernatant fraction is evaporated to dryness using a Savant speed vac. The sediment is reconstituted in assay buffer and subsequently analysed for compound content using the synthetic substrate assay. Briefly, a compound concentration-response curve is constructed for the compound undergoing evaluation. Serial dilutions of the reconstituted plasma extracts are assessed for activity and the amount of compound present in the original plasma sample is calculated using the concentration-response curve taking into account the total plasma dilution factor.
The ability of the compounds of this invention as ex vivo TNFxcex1 inhibitors is assessed in the rat. Briefly, groups of male Wistar Alderley Park (AP) rats (180-210 g) are dosed with compound (6 rats) or drug vehicle (10 rats) by the appropriate route e.g. peroral (p.o.), intraperitoneal (i.p.), subcutaneous (s.c.). Ninety minutes later rats are sacrificed using a rising concentration of CO2 and bled out via the posterior vena cavae into 5 Units of sodium heparin/ml blood. Blood samples are immediately placed on ice and centrifuiged at 2000 rpm for 10 min at 4xc2x0 C. and the harvested plasmas frozen at xe2x88x9220xc2x0 C. for subsequent assay of their effect on TNFxcex1 production by LPS-stimulated human blood. The rat plasma samples are thawed and 175 xcexcl of each sample are added to a set format pattern in a 96 U well plate. Fifty xcexcl of heparinized human blood is then added to each well, mixed and the plate is incubated for 30 min at 37xc2x0 C. (humidified incubator). LPS (25 xcexcl; final concentration 10 xcexcg/ml) is added to the wells and incubation continued for a further 5.5 hours. Control wells are incubated with 25 xcexcl of medium alone. Plates are then centrifuged for 10 min at 2000 rpm and 200 xcexcl of the supernatants are transferred to a 96 well plate and frozen at xe2x88x9220xc2x0 C. for subsequent analysis of TNF concentration by ELISA.
Data analysis by dedicated software calculates for each compound/dose:
Percent inhibition of TNFxcex1=Mean TNFxcex1 (Controls)xe2x88x92Mean TNFxcex1 (Treated)xc3x97100/Mean TNFxcex1 (Controls).
Activity of a compound as an anti-arthritic is tested in the collagen-induced arthritis (CIA) as defined by D. E. Trentham et al., (1977) J. Exp. Med. 146:857. In this model acid soluble native type II collagen causes polyarthritis in rats when administered in Freunds incomplete adjuvant. Similar conditions can be used to induce arthritis in mice and primates.
Activity of a compound as an anti-cancer agent may be assessed essentially as described in I. J. Fidler (1978) Methods in Cancer Research 15:399-439, using for example the B16 cell line (described in B. Hibner et al., Abstract 283 p75 10th NCI-EORTC Symposium, Amsterdam Jun. 16-19 1998).
The invention will now be illustrated but not limited by the following Examples: